Lavelle, S-W; (2016) Investigation into Self-Assembling Microcrystals as an alternative to polysaccharide-based conjugate vaccines. Doctoral thesis , UCL (University College London).

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Abstract

The main virulence factors of encapsulated bacteria such as Haemophilus influenzae type b (Hib) and pneumococcus are their capsular polysaccharides and antibodies against them have been shown to be protective against Hib and pneumococcal invasive diseases. However, polysaccharides are poorly immunogenic and do not induce protective antibody response in children under 2 years when administered on their own. In order to induce a memory B cell response and isotype switching, a protein carrier is usually conjugated to the polysaccharide to stimulate a T cell-dependent response. However, this conjugation process is expensive and the final product is not thermostable. In this project, self-assembling thermostable microcrystals were used to co-immobilise a model capsular polysaccharide (Polyribosylribitol Phosphate; PRP) from Hib and tetanus toxoid (TT) to create a virtual polysaccharide-protein conjugate to mimic the commercially-available Hib conjugate vaccines. Self-assembling microcrystals loaded with both PRP and TT were prepared, and their physico-chemical and immunogenic properties were investigated. In vitro, the factors that influence crystal formation were determined, and antigen loading onto and release from the microcrystals was assessed. The dynamics of the interactions between the crystals and some cells of the immune system were also investigated. In vivo, the crystals were tested in combination with varying buffers, administration routes and adjuvants in an attempt to optimise anti-PRP antibody production. It was found that the crystal morphology and antigen entrapment could be manipulated by preparation conditions. The microcrystal formulation increases cellular uptake of antigens into bone marrow-derived dendritic cells and neutrophils. Despite the use of microcrystals with low level of free PRP, and combinations of different adjuvants, routes, buffers and doses, the microcrystals were not as immunogenic as a commercially-available Hib conjugate vaccine.

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